Mesenchymal Stem Cells
The adult body houses so called stem cells that are capable of dividing many times while also giving rise to daughter cells with specific phenotypical characteristics. Several types of stem cells exist in the body including embryonic stem cells, haematopoietic stem cells and mesenchymal stem cells. Mesenchymal stem cells are able to form mesenchymal tissues such as bone, cartilage, muscle, bone, ligament, fat and bone marrow stroma. FIG. 1 shows a schedule of suggested stepwise transitions from putative mesenchymal stem cells (MSC) to highly differentiated phenotypes. The mesenchymal stem cells are located in bone marrow, around blood vessels, in fat, skin, muscle, bone and other tissues. Their presence contributes to the reparative capacity of these tissues.
Medical use of MSC
Currently, the medical use of MSC is to explore their potential in the regeneration of tissues that the body cannot naturally repair or regenerate when challenged. For this, MSC, are isolated, expanded in culture and stimulated to differentiate into connective tissues such as bone, cartilage, muscle, bone marrow stroma, tendon, fat and others. These tissue-engineered constructs can then be re-introduced into the human body to repair lost or damaged tissue. In another approach MSC can be directly stimulated in vivo to induce the formation of specific tissues in situ.
Having defined MSC as potential “building blocks” for tissue engineering and transplantation, researchers are now searching for better ways to identify, isolate and characterize MSC.
Alpha10
A newly discovered collagen-binding integrin, alpha10beta1, includes the integrin subunit alpha10 (Camper et al., (1998) J. Biol. Chem. 273:20383-20389). The integrin is expressed on chondrocytes and shows a Mr of 160 kDa after reduction when isolated from bovine chondrocytes by collagen type II affinity purification.
Cloning and cDNA sequencing showed that it shares the general structure of other integrin alpha subunits. The predicted amino acid sequence consists of a 1167-amino acid mature protein, including a signal peptide (22 amino acids), a long extracellular domain (1098 amino acids) a transmembrane domain (22 amino acids), and a short cytoplasmic domain (22 amino acids). In contrast to most alpha-integrin subunits, the cytoplasmic domain of alpha10 does not contain the conserved sequence KXGFF(R/K)R (SEQ ID NO: 1). Instead, the predicted amino acid sequence in alpha10 is KLGFFAH (SEQ ID NO: 2). It is suggested that the GFFKR (SEQ ID NO: 3) motif in alpha-chains are important for association of integrin subunits and for transport of the integrin to the plasma membrane (De Melker et al. (1997) Biochem. J. 328:529-537).
The extracellular part contains a 7-fold repeated sequence, an I-domain (199 amino acids) and three putative divalent cation-binding site. Sequence analysis has revealed that the alpha10 subunit is most closely related to the I domain-containing α subunits with the highest identity to alpha1 (37%), alpha2 (35%) and alpha11 (42%).
Alpha11
The alpha11 integrin has recently been identified and cloning and characterisation revealed an I-domain containing, beta1-associated integrin.
The open reading frame of the cDNA encodes a precursor of 1188 amino acids. The predicted mature protein of 1166 amino acids contains 7 conserved FGGAP (SEQ ID NO: 4) repeats, an I-domain with a MIDAS (SEQ ID NO: 5) motif, a short transmembrane region and a unique cytoplasmic domain of 24 amino acids containing the sequence GFFRS (SEQ ID NO: 6).
Alpha11 contains three potential divalent cation binding sites in repeats 5-7. The presence of 22 inserted amino acids in the extracellular stalk portion (amino acids 804-826) distinguishes the alpha11 integrin sequence further from other integrin alpha-chains.
Amino acid sequence comparisons reveal the highest identity (42%) with the alpha10 integrin chain. Immunoprecipitation with antibodies to the alpha11 integrin captured a 145 kDa protein, distinctly larger than the 140 kDa alpha2 integrin chain when analysed by SDS-PAGE under non-reducing conditions.
Isolation and Identification of MSC
The identification of MSC in situ is hampered by the fact that mono-specific and unique molecular probes do not exist. It is therefore necessary to further characterize mesenchymal stem cells to identify probes or combinations of probes that can unequivocally identify mesenchymal stem cells in tissue. Such markers will also be useful for the isolation of mesenchymal stem cells from bone marrow (BM) and blood tissues.
Approximately one cell out of 10.000-100.000 nucleated cells in bone marrow aspirates is expected to be a mesenchymal stem cell. Currently, the main method for the isolation of mesenchymal stem cells from bone marrow is based on their capacity to adhere to plastic culture dishes and form colonies while the majority of bone marrow cells do not adhere and form colonies. These colonies are then further expanded and then induced with defined factors to differentiate into specific mesenchymal tissues. It is not clear, however, whether the mesenchymal stem cells isolated this way are a homogenous population. It will therefore be important to find markers that can be used to identify subclasses of mesenchymal stem cells with specific differentiation potentials.
In U.S. Pat. No. 6,200,606, the isolation of cartilage or bone precursor cells from haematopoietic and non-haematopoietic cells by the use of CD34 as a negative selection marker and the further use of isolated stem cells in bone and cartilage regeneration processes is described. Still, no specific marker for mesenchymal stem cells is identified nor disclosed. The CD34 marker is expressed on early lymphohaematopoietic stem and progenitor cells, small-vessel endothelial cells, embryonic fibroblasts, and some cells in foetal and adult nervous tissue, haematopoietic progenitors derived from foetal yolk sac, embryonic liver, and extra-hepatic embryonic tissues including aorta-associated haematopoietic progenitors in the 5 week human embryo.
Pittenger at al. ((1999) Science 284:143-147) have used a density centrifugation of human bone marrow to isolate human MSC. Cellular markers used to identify the MSC are SH-2, SH-3, CD29, CD44, CD71, CD90, CD106, CD120a, CD124.
Majumdar et al., ((2000) J. Cell. Physiol. 185:98-106) have used CD105 as a marker for enrichment of human MSC from bone marrow.
Denni et al., ((2002) Cells Tissues Organs 170:73-82) have used a marker called Stro-1 to enrich human MSC from bone marrow.
All markers mentioned so far may be used for enrichment of hMSC. Still, they are not exclusive for MSC, the isolated population is heterogenous when enriched using these markers. Monospecific and unique probes for the identification of hMSC do not exist as of today.
Furthermore, markers are needed to monitor the differentiation of mesenchymal stem cells into specific types of mesenchymal cells. This will be especially important when these cells are re-introduced into the human body to replace loss of damaged mesenchymal tissue, such as bone or cartilage.
Finally, the identification of specific cell surface markers for mesenchymal stem cells may be used for their isolation out of a complex mixture of cells by cell sorting techniques such as fluorescence activated cell sorting (FACS).
It is thus highly desirable in the light of the aforementioned problems to identify and isolate MSC, for further use in bone, cartilage, muscle, bone marrow, tendon or connective tissue repair in vivo or in vitro. In this respect, the present invention addresses this needs and interest.